CD36 Knockout Research Articles

CD36 deficiency protects lipopolysaccharide-induced sepsis via inhibiting CerS6-mediated endoplasmic reticulum stress

The type 2 scavenger receptor CD36 functions not only as a long chain fatty acid transporter, but also as a pro-inflammatory mediator. Ceramide is the simple N-acylated form of sphingosine and exerts distinct biological activity depending on its acyl chain length. Six ceramide synthases (CerS) in mammals determine the chain length of ceramide species, and CerS6 mainly produces C16-ceramide. Endotoxin-induced septic shock shows high mortality, but the pathophysiologic role of sphingolipids involved in this process has been hardly investigated. This paper aims to highlight the different role of CerS isoforms in endotoxin-induced inflammatory responses and the regulatory role of CD36 in CerS6 protein degradation with an emphasis as the potential therapeutic candidates in humans. Lipopolysaccharide (LPS), the endotoxin of the Gram-negative bacterial cell wall, was treated to induce endotoxin-induced inflammation both in vitro and in vivo. CerS6-derived C16-ceramide propagated LPS-induced inflammatory responses activating various intracellular signaling pathways, such as mitogen-activated protein kinase and nuclear factor-κB, resulting in the formation of inflammasome complex and pro-inflammatory cytokines. Mechanistically, CerS6-derived C16-ceramide augmented inflammatory responses via endoplasmic reticulum stress, and CerS6 protein stability was regulated by CD36. Finally, CerS6 protein expression and LPS-induced lethality were strikingly reduced in CD36 knockout mice. Collectively, our findings show that CerS6-derived C16-ceramide plays a pivotal role in endotoxin-induced inflammation and suggest CerS6 and its regulator CD36 as possible targets for therapy under life-threatening inflammation such as septic shock.

The fatty acid receptor CD36 promotes macrophage infiltration via p110γ signaling to stimulate metastasis

IntroductionMetabolic regulators are key in controlling immune cell fate in the tumor microenvironment. The accumulation of tumor-associated macrophages (TAMs) in cancer greatly contributes to metastasis and poor outcome. However, the metabolic pathways responsible for TAM accumulation are largely unknown. ObjectiveThis study aims to elucidate the role of the fatty acid translocase CD36 in the regulation of TAM accumulation. MethodsThe immune profile was analyzed in patients with liver metastasis by CIBERSORT. Immunohistostaining of CD68 and CD36 was conducted in clinical specimens from patients with liver metastasis. Myeloid-specific CD36 knockout mice and their littermates were used to establish preclinical liver metastasis models. Subsequently, a series of experiments were used to explore the underlying mechanisms of how CD36 regulates TAM population. ResultsWe found that massive TAM accumulation in patients with liver metastasis is associated with an upregulation of CD36 on TAMs. Liver metastasis is abundantly infiltrated by TAMs that are derived from circulating monocytes, but not tissue-resident macrophages. Myeloid-specific CD36 knockout specifically reduced and inactivated monocyte-differentiated macrophages, resulting in diminished immune suppression and attenuated liver metastasis. The protect effects of CD36 knockout can be abrogated by blockade of macrophage recruitment through CCR2 or the p110γ isoform of PI3K downstream of it. Mechanically, CD36 reprogrammed the lipid metabolism of macrophages, in which sphingolipids were significantly downregulated, that contributed to weakened lipid raft-dependent activation of p110γ. ConclusionCD36 expands TAM population by promoting the recruitment of circulating monocytes through CCL2/CCR2/p110γ signaling. Our findings provide evidence for targeting CD36 as a therapeutic strategy against liver metastasis.

Abstract 3070: Investigating CD36 Oligomerization In Macrophages

Cardiovascular disease (CVD) is the leading cause of mortality worldwide. Atherosclerosis, the main cause of CVD, results from the buildup of cholesterol-filled plaques in artery walls. Accumulation of cholesterol from oxidized low-density lipoprotein (oxLDL) leads to the formation of macrophage foam cells, a process that is mediated by CD36, a scavenger receptor that serves as the primary receptor for oxLDL. The structure of full-length CD36 has not been resolved, nor have the details or role of quaternary structure been fully elucidated. Studies have shown that CD36 has the ability to form homodimers and multimers in some cell types. However, the role of CD36 oligomerization in oxLDL uptake remains unknown. The goal of this study is to investigate CD36 oligomerization in macrophages, a cell type important in the initiation of atherosclerosis. We hypothesize that CD36 oligomerizes in macrophages to facilitate oxLDL uptake. To begin testing this hypothesis, we harvested elicited peritoneal macrophages from wild-type (WT) or CD36 knockout (CD36-KO) mice. We performed crosslinking experiments in which macrophages were incubated with BS3, a membrane-impermeable crosslinker, to stabilize CD36 oligomers. When crosslinked samples were separated by SDS-PAGE electrophoresis, we observed a decrease in monomeric species with increasing crosslinker concentration, however, BS3 also induced aggregation of CD36 rather than oligomer formation. We next turned to Native-PAGE electrophoresis, which lacks detergent entirely, to identify oligomeric species within macrophages, as well as PFO-PAGE electrophoresis, which contains a milder detergent than SDS. Both methods provided evidence of higher-order complexes in WT, but not in CD36-KO macrophages. To further validate our studies, we purified human full-length CD36 using an Sf9 insect cell system and demonstrated that pure CD36 forms homo-oligomers by PFO-PAGE. Together, these experiments provide strong evidence for CD36 oligomerization. Future studies will focus on correlating CD36 oligomerization with its functions in oxLDL binding and uptake. Understanding the higher-order structure of CD36 and how it impacts function will allow us to target CD36 to prevent foam cell formation and atherosclerosis.

Obesity induces an increase in fatty acid uptake in endothelium of visceral adipose arteries without altering CD36 expression

Obesity is a prevalent metabolic condition and major contributor to the development of cardiovascular disease. Arteries of visceral adipose present with significant endothelial dysfunction whereas subcutaneous adipose arteries remain functional in obese humans and diet-induced obese mice. Obesity is also associated with elevated plasma long chain fatty acids (LCFAs), well-established contributors to endothelial dysfunction, which require a receptor to facilitate their uptake. CD36, a scavenger receptor, facilitates LCFA uptake, which when in excess would contribute to endothelial dysfunction. Therefore, we aimed to determine the role of CD36 in arteries ex vivo in both mouse and human subcutaneous and visceral adipose arteries as well as examine the difference in uptake of LCFAs in arteries from both depots. First, to determine if CD36 contributes to obesity-induced endothelial dysfunction in visceral adipose arteries, mesenteric (visceral) and subcutaneous adipose arteries isolated from lean and diet-induced obese WT and CD36 knockout were mounted for pressure myography. As expected, visceral adipose arteries of obese mice exhibited a blunted response to endothelium-dependent stimuli, whereas subcutaneous adipose arteries remained functional. CD36 ablation restored endothelium-dependent vasodilation in visceral arteries of obese mice, suggesting a critical role for CD36 in obesity-induced endothelial dysfunction. We next aimed to determine if obesity alters endothelial CD36 expression using two distinct approaches: flow cytometry and immunofluorescence staining of en face preparations in isolated mouse and/or human arteries. For flow cytometry, arteries were digested and sorted to identify the endothelial cell population (i.e., CD45-CD31+ cells). En face preparations were stained for VE-cadherin to locate the endothelial cell layer via confocal microscopy. In both experiments, an antibody that detects an extracellular epitope to CD36 was used to detect membrane expression. Obesity did not alter the expression of CD36 expression in visceral adipose arteries relative to lean counterpart controls or subcutaneous arteries from obese mice or humans. We next aimed to determine if LCFA uptake was altered by obesity. Arteries were exposed to a fluorescent fatty acid, and internalized endothelial LCFA uptake was assessed at different time points via confocal microscopy. Visceral arteries from obese mice and humans had elevated LCFA uptake relative to respective controls which may be an underlying mediator for the dichotomy in endothelial dysfunction observed in obesity. Ongoing work in our lab is focused on determining if this is indeed the case and, if so, if this process is mediated by endothelial CD36. National Institutes of Health (NIH) Grant P20GM113125-6564 (IF). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 1122: Endothelial Cd36 Mediates Lipid-induced Stiffening Of Aortic Endothelium And Western Diet-induced Barrier Disruption

Introduction: Western diet (WD) associates with elevations in blood lipids. Endothelial cells (EC) uptake lipids through scavenger receptors such as CD36. In our studies we demonstrated that exposure to lipids induces a stiffening response in ECs, with in vivo studies showing that this stiffening is abrogated in mice with a global CD36 knockout. Hypothesis: WD associates with aortic barrier disruption. in vitro studies have demonstrated that EC stiffening correlates with barrier disruption. It is our hypothesis that EC stiffening mediates WD-induced aortic barrier disruption in an endothelial CD36-dependent manner. Methods: To generate a model of endothelial-specific CD36 deficiency, we crossed tamoxifen-inducible Cre mice with an EC-promoter with CD36 fl/fl mice. Cre only mice are employed as controls. At the end of the study window, aortas are excised for assessment of EC stiffening via atomic force microscopy. Separate cohorts are assessed for barrier disruption via quantification of Evans blue dye (EBD) leakage or VE-cadherin junctional thickness. Inhibition of CD36 in vitro was accomplished with SSO. For cell experiments we employed long-chain fatty acids (LCFAs), lipid ligands for CD36. Results: WD induced significant stiffening of the aortic endothelium [p<0.05]. This phenomenon was only observed in male (n=5-6) and not female (n=3-10) mice. EC stiffening associated with aortic barrier disruption as indicated by increased EBD permeation (n=3-8) and increased VE-cadherin thickness (n=3-4) [p<0.05]. Use of an endothelial-specific CD36 deficiency significantly abrogated stiffening and barrier disruption [p<0.05]. Controls demonstrated no effects by tamoxifen (n=2-3). Exposure to 5 μM of palmitic and stearic acids (saturated LCFAs) results in stiffening while exposure to oleic acid (unsaturated LCFA) does not (n=5-12) [p<0.05]. Palmitic acid-induced EC stiffening was shown to be dependent on CD36 (n=5-6) and activation of RhoA protein kinase (n=3-8) [p<0.05]. Conclusions: Our results demonstrate that endothelial CD36 is required for WD-induced EC stiffening and subsequent disruption of the aortic barrier. Furthermore, our results suggest that CD36 mediates EC stiffening through its role as scavenger receptor to saturated LCFAs.

O-GlcNAcylation promotes the progression of nonalcoholic fatty liver disease by upregulating the expression and function of CD36

Background and aimsNonalcoholic fatty liver disease (NAFLD) and its progressive variant, nonalcoholic steatohepatitis (NASH), constitute a burgeoning worldwide epidemic with no FDA-approved pharmacotherapies. The multifunctional immunometabolic receptor, fatty acid translocase CD36 (CD36), plays an important role in the progression of hepatic steatosis. O-GlcNAcylation is a crucial posttranslational modification that mediates the distribution and function of CD36, but its involvement in NAFLD remains poorly understood. MethodsO-GlcNAcylation and CD36 expression were evaluated in human liver tissues obtained from NASH patients and normal control. Mice with hepatocyte-specific CD36 knockout were administered adeno-associated viral vectors expressing wild-type CD36 (WT-CD36) or CD36 O-GlcNAcylation site mutants (S468A&T470A-CD36) and were provided with a high-fat/high-cholesterol (HFHC) diet for 3 months. RT–qPCR analysis, immunoblotting, dual-luciferase reporter assays, chromatin immunoprecipitation, and coimmunoprecipitation were performed to explore the mechanisms by which O-GlcNAcylation regulates CD36 expression. Membrane protein extraction, immunofluorescence analysis, site-directed mutagenesis, and fatty acid uptake assays were conducted to elucidate the impact of O-GlcNAcylation on CD36 function. ResultsO-GlcNAcylation and CD36 expression were significantly increased in patients with NASH, mouse models of NASH, and palmitic acid-stimulated hepatocytes. Mechanistically, the increase in O-GlcNAcylation facilitated the transcription of CD36 via the NF-κB signalling pathway and stabilized the CD36 protein by inhibiting its ubiquitination, thereby promoting CD36 expression. On the other hand, O-GlcNAcylation facilitated the membrane localization of CD36, fatty acid uptake, and lipid accumulation. However, site-directed mutagenesis of residues S468 and T470 of CD36 reversed these effects. Furthermore, compared with their WT-CD36 counterparts, HFHC-fed S468A&T470A-CD36 mice exhibited decreases in systemic insulin resistance, steatosis severity, inflammation and fibrosis. Pharmacological inhibition of O-GlcNAcylation and CD36 also mitigated the progression of NASH. ConclusionsO-GlcNAcylation promotes the progression of NAFLD by upregulating CD36 expression and function. Inhibition of CD36 O-GlcNAcylation protects against NASH, highlighting a potentially effective therapeutic approach for individuals with NASH.

Systemic loss of CD36 aggravates NAFLD-related HCC through MEK1/2-ERK1/2 signaling pathway

Background & aimsCD36, a membrane protein widely present in various tissues, is crucial role in regulating energy metabolism. The rise of HCC as a notable outcome of NAFLD is becoming more apparent. Patients with hereditary CD36 deficiency are at increased risk of NAFLD. However, the impact of CD36 deficiency on NAFLD-HCC remains unclear. MethodsGlobal CD36 knockout mice (CD36KO) and wild type mice (WT) were induced to establish NAFLD-HCC model by N-nitrosodiethylamine (DEN) plus high fat diet (HFD). Transcriptomics was employed to examine genes that were expressed differentially. ResultsCompared to WT mice, CD36KO mice showed more severe HFD-induced liver issues and increased tumor malignancy. The MEK1/2-ERK1/2 pathway activation was detected in the liver tissues of CD36KO mice using RNA sequencing and Western blot analysis. ConclusionSystemic loss of CD36 leaded to the advancement of NAFLD to HCC by causing lipid disorders and metabolic inflammation, a process that involves the activation of MAPK signaling pathway. We found that CD36 contributes significantly to the maintenance of metabolic homeostasis in NAFLD-HCC.

CNP Ameliorates Macrophage Inflammatory Response and Atherosclerosis.

CNP (C-type natriuretic peptide), an endogenous short peptide in the natriuretic peptide family, has emerged as an important regulator to govern vascular homeostasis. However, its role in the development of atherosclerosis remains unclear. This study aimed to investigate the impact of CNP on the progression of atherosclerotic plaques and elucidate its underlying mechanisms. Plasma CNP levels were measured in patients with acute coronary syndrome. The potential atheroprotective role of CNP was evaluated in apolipoprotein E-deficient (ApoE-/-) mice through CNP supplementation via osmotic pumps, genetic overexpression, or LCZ696 administration. Various functional experiments involving CNP treatment were performed on primary macrophages derived from wild-type and CD36 (cluster of differentiation 36) knockout mice. Proteomics and multiple biochemical analyses were conducted to unravel the underlying mechanism. We observed a negative correlation between plasma CNP concentration and the burden of coronary atherosclerosis in patients. In early atherosclerotic plaques, CNP predominantly accumulated in macrophages but significantly decreased in advanced plaques. Supplementing CNP via osmotic pumps or genetic overexpression ameliorated atherosclerotic plaque formation and enhanced plaque stability in ApoE-/- mice. CNP promoted an anti-inflammatory macrophage phenotype and efferocytosis and reduced foam cell formation and necroptosis. Mechanistically, we found that CNP could accelerate HIF-1α (hypoxia-inducible factor 1-alpha) degradation in macrophages by enhancing the interaction between PHD (prolyl hydroxylase domain-containing protein) 2 and HIF-1α. Furthermore, we observed that CD36 bound to CNP and mediated its endocytosis in macrophages. Moreover, we demonstrated that the administration of LCZ696, an orally bioavailable drug recently approved for treating chronic heart failure with reduced ejection fraction, could amplify the bioactivity of CNP and ameliorate atherosclerotic plaque formation. Our study reveals that CNP enhanced plaque stability and alleviated macrophage inflammatory responses by promoting HIF-1α degradation, suggesting a novel atheroprotective role of CNP. Enhancing CNP bioactivity may offer a novel pharmacological strategy for treating related diseases.

CD36 inhibition reduces non-small-cell lung cancer development through AKT-mTOR pathway

Lung cancer is the most common cause of cancer-related deaths worldwide and is caused by multiple factors, including high-fat diet (HFD). CD36, a fatty acid receptor, is closely associated with metabolism-related diseases, including cardiovascular disease and cancer. However, the role of CD36 in HFD-accelerated non-small-cell lung cancer (NSCLC) is unclear. In vivo, we fed C57BL/6J wild-type (WT) and CD36 knockout (CD36−/−) mice normal chow or HFD in the presence or absence of pitavastatin 2 weeks before subcutaneous injection of LLC1 cells. In vitro, A549 and NCI-H520 cells were treated with free fatty acids (FFAs) to mimic HFD situation for exploration the underlying mechanisms. We found that HFD promoted LLC1 tumor growth in vivo and that FFAs increased cell proliferation and migration in A549 and NCI-H520 cells. The enhanced cell or tumor growth was inhibited by the lipid-lowering agent pitavastatin, which reduced lipid accumulation. More importantly, we found that plasma soluble CD36 (sCD36) levels were higher in NSCLC patients than those in healthy ones. Compared to that in WT mice, the proliferation of LLC1 cells in CD36−/− mice was largely suppressed, which was further repressed by pitavastatin in HFD group. At the molecular level, we found that CD36 inhibition, either with pitavastatin or plasmid, reduced proliferation- and migration-related protein expression through the AKT/mTOR pathway. Taken together, we demonstrate that inhibition of CD36 expression by pitavastatin or other inhibitors may be a viable strategy for NSCLC treatment.Graphical abstract1) Pitavastatin reduces NSCLC progression by inhibiting CD36.2) Inhibition of CD36 can improve HFD- or FFA-induced NSCLC.3) AKT/mTOR pathway is involved in CD36-regulated NSCLC.4) Inhibition of CD36 by pitavastatin or other inhibitors may be a strategy for NSCLC treatment.

CD36 deletion ameliorates diabetic kidney disease by restoring fatty acid oxidation and improving mitochondrial function

Renal tubular epithelial cells (TECs) are vulnerable to mitochondrial dysregulation, which is an integral part of diabetic kidney disease (DKD). We found that CD36 knockout ameliorated mitochondrial dysfunction and diabetic kidney injury in mice, improved renal function, glomerular hypertrophy, tubular injury, tubulointerstitial fibrosis, and kidney cell apoptosis. Furthermore, CD36 knockout conferred protection against diabetes-induced mitochondrial dysfunction and restored renal tubular cells and mitochondrial morphology. CD36 knockout also restored mitochondrial fatty acid oxidation (FAO) and enhanced FAO-associated respiration in diabetic TECs. CD36 was found to alter cellular metabolic pathways in diabetic kidneys partly via PDK4 the –AMPK axis inactivation. Because CD36 protects against DKD by improving mitochondrial function and restoring FAO, it can serve as a potential therapeutic target.

Abstract P1087: Cd36-retinoic Acid-PPARδ Signaling Axis Modulates Cardiomyocytes’ Ability To Proliferate In Newly Born Neonatal Mice

Background: A pertinent question is why only a subset of fetal/neonatal cardiomyocytes (CMs) spontaneously proliferate. Using single-cell RNAseq, we previously showed a specific population of CMs that is more responsive to cell cycle induction (Abouleisa et al., Circulation, 2022). This population is characterized by expression of the transmembrane scavenger receptor, CD36 (fatty acid (FA) internalization receptor). Here, we aim to investigate the mechanism by which CD36 modulates CMs decision to enter into the cell cycle in neonatal P1 CMs. Methods and Results: Our single-cell RNAseq from primary CMs isolated from P1 hearts demonstrated that CD36 is specifically expressed in the spontaneously proliferating CMs and the CMs that respond to the exogenous cell cycle stimulation. CMs isolated from P1 CD36 knockout mice (CD36 KO ) showed 50% less proliferation capacity compared to WT P1 CMs. Interestingly, CD36 KO mice are born with smaller hearts which contain fewer CMs compared with their WT littermates. Furthermore, compared with WT littermates, which completely regenerate the heart apex following apical resection at P1, CD36 KO showed minimal regenerative capacity. Surprisingly, CD36 KO CMs didn’t show significant disruption in FA oxidation or glycolysis; however, bulk RNAseq data from the P1 CD36 KO hearts showed significant downregulation in the expression of retinoic acid (RA)-dependent signaling genes (e.g., PPARδ and RXRa) and a significant increase in PPARα expression. Confirming a causal role of CD36 in CM proliferation, we found that Cd36 is crucial in regulating the entry and interactions of RA or retinoid precursors in CMs. The CD36 + FACS-sorted CMs express significantly higher levels of PPARδ, RXRa, and FABP5 and lower levels of PPARα, which are crucial in regulating the response to cell proliferation. Furthermore, small molecule activation of PPARδ doubled cell cycle entry in P1 CMs and completely restored proliferation in CD36 KO CMs. These data suggest that CD36 is essential to prime CMs proliferation during development by regulating RA/PPARδ/RXR transcriptional signaling. Conclusions: These findings suggest that the CD36-RA-PPARδ signaling axis modulates the CMs’ ability of P1 to spontaneously enter the cell cycle.

Expression profiling and antibacterial analysis of cd36 in mandarin fish, Siniperca chuatsi.

Cd36 is classified as a class B scavenger receptor and has also been identified as a pattern recognition receptor. In this study, we investigated the genomic structure and molecular characteristics of cd36 in mandarin fish (Siniperca chuatsi), examined its tissue distribution, and evaluated its antibacterial activity. Genomic structure analysis showed that Sccd36 consists of 12 exons and 11 introns. Sequencing analysis confirmed that the open reading frame of Sccd36 contains 1410 bp, encoding 469 amino acids. Sccd36 is deeply conserved with other vertebrates in terms of genomic structure, gene loci and molecular evolution, and the feature of two transmembrane was observed in ScCd36 through structural prediction. Sccd36 was constitutively expressed in all tissues tested, with the strongest expression in the intestine, followed by the heart and the kidney. Dramatic changes of Sccd36 mRNA were detected in mucosal tissues, including the intestine, gill and skin, when stimulated by the microbial ligands lipopolysaccharide and lipoteichoic acid. In addition, ScCd36 was identified as having strong binding ability to microbial ligands and antibacterial activity against the gram-negative bacteria Aeromonas hydrophila and the gram-positive bacteria Streptococcus lactis. Furthermore, we verified that the genetic ablation of cd36 impaired the resistance of fish to bacterial challenge by using zebrafish cd36 knockout line. In conclusion, our findings suggest that ScCd36 plays a crucial role in the innate immune response of mandarin fish against bacterial infections. This also sets the stage for further exploration into the antibacterial function of Cd36 in lower vertebrate species.

Aging-Related Accumulation of Truncated Oxidized Phospholipids Augments Infectious Lung Injury and Endothelial Dysfunction via Cluster of Differentiation 36-Dependent Mechanism.

Truncated phospholipid oxidation products (Tr-OxPL) increase in blood circulation with aging; however, their role in the severity of vascular dysfunction and bacterial lung injury in aging groups remains poorly understood. We investigated the effects of six Tr-OxPL species: KOdiA-PC, POVPC, PONPC, PGPC, Paz-PC, and Lyso-PC on endothelial dysfunction and lung inflammation caused by heat-killed Staphylococcus aureus (HKSA) in young (aged 2-4 months) and old (aged 12-18 months) mice, organotypic culture of precisely cut lung slices, and endothelial cells (mLEC) isolated from young and old mice. HKSA and Tr-OxPL combination caused a higher degree of vascular leak, the accumulation of inflammatory cells and protein in bronchoalveolar lavage, and inflammatory gene expression in old mice lungs. HKSA caused a greater magnitude of inflammatory gene activation in cell and ex vivo cultures from old mice, which was further augmented by Tr-OxPLs. L37pA peptide targeting CD36 receptor attenuated Tr-OxPL-induced endothelial cell permeability in young and old mLEC and ameliorated KOdiA-PC-induced vascular leak and lung inflammation in vivo. Finally, CD36 knockout mice showed better resistance to KOdiA-PC-induced lung injury in both age groups. These results demonstrate the aging-dependent vulnerability of pulmonary vasculature to elevated Tr-OxPL, which exacerbates bacterial lung injury. CD36 inhibition is a promising therapeutic approach for improving pneumonia outcomes in aging population.

Hepatocyte CD36 modulates UBQLN1-mediated proteasomal degradation of autophagic SNARE proteins contributing to septic liver injury

ABSTRACT Macroautophagy/autophagy plays a protective role in sepsis-induced liver injury. As a member of class B scavenger receptors, CD36 plays important roles in various disorders, such as atherosclerosis and fatty liver disease. Here we found that the expression of CD36 in hepatocytes was increased in patients and a mouse model with sepsis, accompanied by impaired autophagy flux. Furthermore, hepatocyte cd36 knockout (cd36-HKO) markedly improved liver injury and the impairment of autophagosome-lysosome fusion in lipopolysaccharide (LPS)-induced septic mice. Ubqln1 (ubiquilin 1) overexpression (OE) in hepatocyte blocked the protective effect of cd36-HKO on LPS-induced liver injury in mice. Mechanistically, with LPS stimulation, CD36 on the plasma membrane was depalmitoylated and distributed to the lysosome, where CD36 acted as a bridge molecule linking UBQLN1 to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and hence promoting the proteasomal degradation of SNARE proteins, resulting in fusion impairment. Overall, our data reveal that CD36 is essential for modulating the proteasomal degradation of autophagic SNARE proteins in a UBQLN1-dependent manner. Targeting CD36 in hepatocytes is effective for improving autophagic flux in sepsis and therefore represents a promising therapeutic strategy for clinical treatment of septic liver injury. Abbreviations: AAV8: adeno-associated virus 8; AOSC: acute obstructive suppurative cholangitis; ATP1A1: ATPase, Na+/K+ transporting, alpha 1 polypeptide; CASP3: caspase 3; CASP8: caspase 8; CCL2: chemokine (C-C motif) ligand 2; cd36-HKO: hepatocyte-specific cd36 knockout; Co-IP: co-immunoprecipitation; CQ: chloroquine; Cys: cysteine; GOT1: glutamic-oxaloacetic transaminase 1, soluble; GPT: glutamic–pyruvic transaminase, soluble; IL1B: interleukin 1 beta; IL6: interleukin 6; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LDH, lactate dehydrogenase; LPS: lipopolysaccharide; LYPLA1: lysophospholipase 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OE: overexpression; qPCR: quantitative polymerase chain reaction; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TNF: tumor necrosis factor; TRIM: tripartite motif-containing; UBA: ubiquitin-associated; UBL: ubiquitin-like; UBQLN: ubiquilin; VAMP8: vesicle associated membrane protein 8; WT: wild-type.

Abstract 1732: Functional characterization of the impact of CD36 deletion on normal hematopoiesis and the leukemia microenvironment

Abstract CD36 has been identified as a potential therapeutic target both in the leukemic cells and in the tumor immune microenvironment. CD36 also plays a role in lipid metabolism of cancer associated T-cells leading to impaired cytotoxic CD8+ T-cell and enhanced Treg cell function. In acute myeloid leukemia (AML), we found that APOC2 acts with CD36 to promote leukemia growth by activating the LYN-ERK signaling. To establish CD36 as a viable therapeutic target in AML, we recently investigated whether targeting CD36 has any detrimental impact on normal hematopoietic cells. We compared B6.129S1-Cd36tm1Mfe/J Cd36 knockout (Cd36-KO) with C57BL/6J control (WT) mice (N=6 per group). Cd36-KO mice have normal blood counts except for lower red blood cells count (5.85 vs 6.93 (x 106/μL), P = 0.0332), hemoglobin (10.03 vs 11.35 (gr/dL), P = 0.0109), and hematocrit (31.67 vs 35.48 (%), P = 0.02) than WT mice, yet these values remained within the normal range. Cd36-KO mice exhibited similar spleen T cell phenotypes compared with the WT mice and expanded similarly in vitro in the presence IL2 and PHA. The in vitro expansion of hematopoietic stem and progenitor cells (HSPC) in HSPC expansion media containing SCF and TPO cytokines was also similar between Cd36-KO mice and WT mice. However, mouse colony forming cell assay using methylcellulose-based media showed that the HSPC of Cd36-KO mice resulted in 40% fewer colonies than WT mouse HSPC cells (P = 0.0003). However, when HSPC obtained from Cd36-KO and WT mice were transplanted in CD45.1 mice, their percentage of engraftment were not significantly different in the bone marrow (BM: KO vs WT: 64.92% vs 63.45%, P > 0.9999), spleen (40.88% vs 40.05%, P = 0.8413), and blood (83.47% vs 82.88%, P > 0.9999). To assess whether CD36 in the leukemia microenvironment has an impact on leukemia growth and progression, we injected 5 x 106 FLT3-ITD/MLL-PTD mouse leukemic cells via tail vein in either Cd36-KO mice (N = 5) or WT mice (N = 6) and compared the percentage of engraftment in their tissues. Cd36-KO mice exhibited similar leukemia engraftment in the BM (88.92% vs 86.38%, P = 0.0497), spleen (76.48% vs 68.10%, P = 0.1627), liver (16.12% vs 6.70%, P = 0.2521), and blood (16.33% vs 0.96%, P = 0.2504) compared with the WT group. Cd36-KO and WT mice developed similar leukemia burden when less aggressive models are used with the engraftment of 1 x 106 FLT3-ITD/MLL-PTD mouse leukemic cells. Altogether, our preliminary data suggest that loss of Cd36 may influence the hematopoietic stem cell and erythropoiesis yet have limited detrimental overall impact on normal hematopoiesis and leukemia microenvironment. Therefore, targeting CD36 in leukemic cells presents limited toxicity on normal hematopoiesis and therapeutic approaches to target this receptor present safe strategies to treat AML. Citation Format: Yiting Meng, Mateusz Pospiech, Atham Ali, Ritu Chandwani, Sandra Onyemaechi, George Yaghmour, Houda Alachkar. Functional characterization of the impact of CD36 deletion on normal hematopoiesis and the leukemia microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1732.

CD36 regulates diurnal glucose metabolism and hepatic clock to maintain glucose homeostasis in mice

The mammalian circadian clock and glucose metabolism are highly interconnected, and disruption of this coupling is associated with multiple negative health outcomes. Liver is the major source of endogenous glucose production and liver clock is one of the most vital peripheral clock systems. We demonstrate that fatty acid translocase (CD36) is expressed rhythmically in mouse liver and autonomously modulates the diurnal oscillations of liver clock and glucose homeostasis. CD36 knockout in hepatocytes inhibits the relay of insulin signaling and provokes FoxO1 nuclear shuttling, consequently increasing Per1 nuclear expression. Moreover, FoxO1 can activate the central clock gene Per1 at the transcriptional level. These changes lead to a disrupted clock oscillation and behavioral rhythm. Our study first reveal that CD36 is a key regulator of the circadian oscillator and its deficiency may cause liver clock disruption, which aggravates the imbalance of glucose homeostasis and contribute to augmentation and progression of metabolic disease.

Abstract TP242: The Absence Of CD36 Reduced Kras G12V -induced Brain Arteriovenous Malformations In Mice.

Introduction: Brain arteriovenous malformations (bAVMs) are a major risk factor for cerebral hemorrhages in young patients. Recent clinical studies have identified somatic activating KRAS mutations in human bAVMs (~76%), and we confirmed the causal role of endothelial KRAS G12V mutation in bAVM development using a mouse model. However, the pathomechanisms underlying bAVM pathology remain to be determined. CD36 is highly expressed in microglia/macrophages (MMs) and microvascular endothelial cells (ECs) and plays various roles in inflammation and angiogenesis, closely related to vascular development and functions. In this study, we determined if CD36 is involved in bAVM pathophysiology. Methods: Mice were injected with either AAV-BR1 carrying GFP (control) or KRAS G12V . We determined CD36 expression in the control or bAVM tissues dissected from the mouse brains. We also determined CD36 levels in the in vitro cultured ECs overexpressing KRAS G12V . Finally, we determined bAVM numbers and size in CD36 knock-out (ko) mice injected with AAV-BR1-KRAS G12V . Results: We found that gene expression of CD36 and the ligands, thrombospondin-1 and -2 were significantly increased in the KRAS G12V -induced bAVMs. However, the CD36 levels were slightly but significantly reduced in KRAS G12V transfected ECs. In the CD36 ko mice, we found that the number of large bAVMs was significantly reduced compared to the control (C57BL/6). Conclusion: Our results indicate that CD36 expression is dysregulated in KRAS G12V -induced bAVMs and contributes to bAVM development. As CD36 has distinctive roles depending on cell types, the role of CD36 in each cell type (e.g., MMs or ECs) on bAVM pathology should be further determined.

Loss of Cd36 Expression Has Limited Impact on Mouse Normal Hematopoiesis

CD36 has been identified as a potential therapeutic target both in the leukemic cells and in tumor immune microenvironment. In acute myeloid leukemia, we recently found that APOC2 acts with CD36 to promote acute myeloid leukemia (AML) growth by activating the LYN-ERK signaling. CD36 also plays a role in lipid metabolism of cancer associated T-cells leading to impaired cytotoxic CD8+ T-cell and enhanced Treg cell function. CD36 is also present on various types of normal hematopoietic cells, including monocytes, macrophages, endothelial cells, and platelets and contribute to their normal functions. Whether targeting CD36 presents unwanted consequences on normal hematopoiesis remains unknown. Here we analyzed mouse RNA-Seq data (BloodSpot, GSE60101) and found that Cd36 expression is low in the hematopoietic stem cells (HSC) and increases significantly as they differentiate to mature cells (115.4-fold in erythrocytes, 61.8-fold in NK cells, 32.9-fold in B cells, P < 0.001). We also observed differential expression patterns of CD36 RNA between human and mouse. The haemopedia human RNAseq data revealed high level of CD36 expression in dendritic cell lineage, macrophage/monocytes, and NK cells. While haemopedia mouse RNAseq data showed higher Cd36 expression in progenitor cells, erythrocytes, mast cells, basophils, and B cells. To investigate the effect of loss of Cd36 in normal hematopoiesis, we compared B6.129S1-Cd36tm1Mfe/J Cd36 knockout (KO) with C57BL/6J control (WT) mice (N=6 per group). The mice strains were confirmed by genotyping. In bone marrow (BM), spleen, and liver cells, the KO mice had reduced Cd36 mRNA level (~46-72%, P < 0.05), and decreased Cd36 surface expression (~20-40% Cd36+ population, P < 0.05) compared with WT mice. Differential blood count and hematological analysis revealed lower red blood cells count (5.85 vs 6.93 (x 106/uL), P = 0.0332), hemoglobin (10.03 vs 11.35 (gr/dL), P = 0.0109), and hematocrit (31.67 vs 35.48 (%), P = 0.02) in KO mice than WT mice, yet these values remained within the normal range. On the other hand, the count of white blood cells (1.47 vs 2.06 (x 103/uL), P = 0.2461), neutrophil (5.77 vs 6.10 (%), P = 0.8750), lymphocytes (81.55 vs 86.48 (%), P = 0.1509), monocytes (5.27 vs 3.23 (%), P = 0.0848), eosinophils (7.40 vs 4.08 (%), P = 0.3223), and platelets (230.67 vs 280.17 (x 1000/uL), P = 0.6779) were not significantly different between Cd36 KO and WT mice. Cd36 KO mice also exhibited similar T cell phenotype in the spleen compared with the WT mice: (% lymphocytes: 72.13% vs 67.97%, P = 0.7857; % CD3+: 29.80% vs 29.10%, P = 0.6982; % CD4+: 57.02% vs 54.52%, P = 0.0901; % CD8+: 32.12% vs 33.70%, P = 0.1246; and % CD25+: 12.03% vs 21.03%, P = 0.9920). We also assessed the in vitro expansion of hematopoietic stem and progenitor cells (HSPC) in HSC expansion media containing SCF and TPO cytokines. The expansion of HSPC was not significantly different (P = 0.2461) between WT and KO mice (Day 10 to Day 5 fold change: 1.65-fold, P = 0.0187 in WT and 1.92-fold, P = 0.0049 in KO). Consistently, the MFI of CFSE labeled HSPC was decreased by 35% (P = 0.0047) in WT and 32% (P = 0.0006) in KO on day 9 relative to day 1 (P = 0.8292 between KO and WT). However, mouse colony forming cell assay using methylcellulose-based media showed that the HSPC of KO mice resulted in less colonies compared with WT mice (WT vs KO = 44 vs 26, P = 0.0108). We also assessed T cells proliferation in CD36 KO mice by in vitro culturing of mouse splenocytes with T cell expansion media containing IL2 and PHA. The number of viable T cells was 1.94-fold (P < 0.0001) in WT mice and 2.24-fold (P < 0.0001) in KO mice on day 10 relative to day 5 (P = 0.2519 comparing WT vs KO). In addition, the cell trace dye MFI was decreased by 67% (P = 0.0002) in WT splenocytes and 59% (P = 0.0030) in KO solenocytes (P = 0.2879 comparing WT vs KO). To assess the leukemia burden in KO mice, we injected 5 million FLT3-ITD/MLL-PTD mice leukemic cells via tail vein in either Cd36 KO mice (N = 5) or WT mice (N = 6) and compared the percentage of engraftment in their tissues. Cd36 KO mice exhibited similar leukemia engraftment in the BM (88.92% vs 86.38%, P = 0.0497), spleen (76.48% vs 68.10%, P = 0.1627), liver (16.12% vs 6.70%, P = 0.2521), and blood (16.33% vs 0.96%, P = 0.2504) compared with the WT group. Altogether, our preliminary data suggest that Cd36-KO has limited impact on normal hematopoietic cells.

Abstract 391: Dissecting The Role Of Macrophage Cd36 In Resolution Of Inflammation

Monocytes and macrophages are implicated in cardiovascular disease, including atherosclerotic plaque progression and regression. The scavenger receptor CD36, which is highly expressed in macrophages, mediates lipid uptake into macrophages and promotes clearance of apoptotic and dead cells. Moreover, CD36 as multi-ligand scavenger receptor binds both fatty acids and lipoproteins, which differ in their effects on macrophage phenotype. Some in vitro experiments have reported that CD36-mediated uptake of lipids convert macrophages to an inflammatory phenotype, whereas other studies show that lipid uptake converts macrophages to an alternatively activated state. To determine the role of this receptor in vivo where cells are exposed to both lipoproteins and free fatty acids, we created macrophage-specific CD36 knockout mice. Using zymosan-induced peritonitis to challenge immune cells, we found a broad heterogeneity of resident and infiltrated immune cells, primarily macrophages, in the peritoneum 72h after the initial stimuli. Macrophage CD36 deficient cells had increased expression of pathways related to innate immunity, antigen presentation and oxidative phosphorylation; whereas pathways related to efferocytosis adaptive immunity and B cell activation were downregulated. Gene signatures related to pro-inflammatory features were evident only in CD36 deficient macrophages. Macrophage efferocytosis capacity and B cell antibody production did not differ between control and CD36 depleted mice. Our data show that CD36 expression allows macrophages to convert to a less inflammatory state.

IL-6 promotes chemoresistance via upregulating CD36 mediated fatty acids uptake in acute myeloid leukemia

Chemoresistance contributes to poor survival and high relapse risk in acute myeloid leukemia (AML). As a pro-inflammatory cytokine, interleukin-6 (IL-6) plays a vital role in the chemoresistance of malignancies. However, the underlying mechanisms of chemoresistance in AML have not been widely studied. Lipid metabolism, which contributes to chemoresistance in AML, is enhanced by IL-6 in skeletal muscle cells. We hypothesized that IL-6 promotes the chemoresistance of AML by promoting lipid metabolism. Based on the positive correlation between IL-6 receptor expression and the cellular response to exogenous IL-6, we performed Gene Ontology analysis of a dataset consisting the information of 151 AML patients from The Cancer Genome Atlas. We found that lipid transport-associated genes were upregulated in the high IL-6 receptor expression group. Additionally, IL-6 promoted fatty acid (FA) uptake in both AML cell lines and primary AML cells. Inhibition of FA uptake by sulfo-N-succinimidyl oleate repressed IL-6-induced chemoresistance. Western blotting, quantitative polymerase chain reaction, and chromatin immunoprecipitation assays indicated that IL-6 promoted CD36 expression at both the mRNA and protein levels through stat3 signaling. Knockout of CD36 or stat3 repressed IL-6-induced FA uptake and chemoresistance. Furthermore, in five human AML samples, we validated that compared to CD36−cells, CD36+ primary AML cells were less sensitive to cytosine arabinoside (Ara-c) and that blockade of CD36 re-sensitized CD36+ AML cells to Ara-c. Mice injected with CD36 knockout cells followed by treatment with Ara-c showed markedly decreased leukemia burden and prolonged survival in vivo. Finally, treatment with the CD36 antibody in combination with Ara-c exhibited synergistic effects in vivo. In conclusion, IL-6 promotes chemoresistance in AML through the stat3/CD36-mediated FA uptake. Blockade of CD36 improved the effect of Ara-c, representing a promising strategy for AML therapy.